CAS number: 9025-71-2
EC number: 3.1.1.20
Tannase is a key enzyme in the degradation of gallotannins and ellagicitannins, two types of hydrolysable tannins.
Specifically, tannase catalyzes the hydrolysis of ester and depside bonds of hydrolysable tannins to release glucose and gallic or ellagic acid.
Tannase has an enzyme commission number (EC) of 3.1.1.20.
Tannase has this specific EC number as it belongs to the family of hydrolases, specifically those acting on carboxylic ester bonds.
The systematic name of Tannase class is tannin acylhydrolase.
Other names of Tannase in common use include tannase S, and tannin acetylhydrolase.
Features of Tannase:
Tannase decomposes the tea gallated polyphenols into gallic acid and polyphenols to prevent combining with caffeine which is the cause of tea turbidity.
Tannase can clear any kinds of teas but does not change the taste.
Applications of Tannase:
Tannase participates in fruit ripening by breaking the ester bonds of glucose with chebulinic, gallic and hexahydrophenic acid.
Tannase is also used in the food, feed, beverages, pharmaceutical, and chemical industries to produce gallic acid, instant tea, coffee flavored refreshing drinks and acron wines.
In addition, tannase is used to clarify beer and juice, improve the flavor of the wine and make animal feed.
In the chemical industry, tannase can be used to analytical probe preparation, determine the structure of naturally occurring gallic acid esters, detect cancer cells, and treat tannins-containing wastewater in the olive oil and leather industries.
Tannase has two known domains and one known active site.
Tannase can be found in plants, bacteria, and fungi and has different purposes depending on the organism it is found in.
Tannase also has many purposes for human use.
The production of gallic acid is important in the pharmaceutical industry as it's needed to create trimethoprim, an antibacterial drug.
Tannase also has many applications in the food and beverage industry.
Specifically, Tannase is used to make food and drinks taste better, either by removing turbidity from juices or wines, or removing the bitter taste of tannins in some food and drinks, such as acorn wine.
Additionally, because tannase can break ester bonds of glucose with various acids (chebulinic, gallic, and hexahydrophenic), it can be used in the process of fruit ripening.
Mechanism of Tannase:
In enzymology, a tannase is an enzyme that catalyzes the chemical reaction.
Thus, the two substrates of Tannase are digallate and H2O, whereas its product is gallate.
In addition to catalyzing the hydrolysis of the central ester bond between the two aromatic rings of digallate (depsidase activity), tannase may also have an esterase activity (hydrolysis of terminal ester functional groups that are attached to only one of the two aromatic rings).
Digallate is the conjugate base of digallic acid, but are often used synonymously.
Similarly, gallate and gallic acid are used interchangeably.
Both digallic and gallic acid are organic acids that are seen in gallotannins and are usually esterified to a glucose molecule.
In other words, tannins (which contain digallate/digallic acid) are the natural substrate of tannase.
When tannins, specifically gallotannins, are broken down by tannase through the hydrolysis of ester bonds, gallic acid and glucose are formed.
Structure of Tannase:
The crystal structure of tannase varies slightly depending on the strain being observed, in this case we are looking at the tannase SN35N strain produced in Lactobacillus plantarum.
On average, its molecular weight is in the range of 50-320 kDa.
Domains of Tannase:
Tannase from Lactobacillus plantarum has 489 amino acid residues and two domains.
The two domains of tannase are called the α/β-hydrolase domain and the lid domain.
The α/β-hydrolase domain consists of residues 4-204 and 396-469, and is composed of two nine-stranded β-sheets surrounded by four α-helices on one side and two α-helices on the other side.
Conversely, the lid domain consists of residues 205–395 and is composed of seven α-helices and two β-sheets.
Active sites:
There is one known active site in tannase found in the SN35N strain.
The crystal structure shows there is a tunnel formed by two opposing domains that can fit the various substrates needed for tannase to hydrolyze.
This active site is referred to as the Ser163 active site and is located in the α/β-hydrolase domain.
In this active site Ser163, Asp419, and His451 residues form a catalytic triad.
If any one of these residues are mutated in the catalytic triad, tannase activity almost always stops.
Structure and function of Tannase:
One way in which the structure of tannase is tied with its function involves a loop structure, called the flap.
The flap connects β8 and β9 sheets and is located under the catalytic triad.
As a result of weak electron densities, this structure is very flexible.
Due to Tannases flexibility, the flap is better able to guide the substrate in entering the enzyme and helps to strengthen the overall binding of the complex by forming additional interactions with other parts of the substrate.
Official Full Name:
Tannase
Background:
Tannase catalyzes the hydrolysis of tannic acid to produce gallic acid and glucose.
Synonyms:
tannase; 9025-71-2; Tannin acyl Hydrolase
Functions of Tannase:
Plants:
Tannase functions differently in the cell depending on the organism being observed.
In many plants, tannase is used to produce tannins, which are found in leaves, wood, and bark.
The production of tannins in plants is essential for defense against herbivory, as they cause a strong unpalatable flavor.
Tannins are considered secondary metabolites in plants.
Therefore, their production by tannase plays no direct role in plant primary metabolism.
Microorganisms:
On the other hand, tannase serves a different purpose in many microorganisms.
In the cell, tannase is a key enzyme in the degradation of gallotannins.
This is important, because some microorganisms use tannase to breakdown hydrolysable tannins, such as gallotannins, to form glucose and gallic acid.
These byproducts are created from the hydroxylation of the aromatic nucleus of the tannin, followed by ring cleavage.
Glucose and gallic acid can then be readily converted to metabolites (i.e. pyruvate, succinate, and acetyl coenzyme A) that can be used in the Krebs cycle.
Specific microorganisms that utilize tannase in this way include Pseudomonas species.
Species distribution:
Tannase is present in a diverse group of microorganisms, including rumen bacteria.
Many other bacterial species have been found to produce tannase by being isolated from different types of media such as soil, wastewater, compost, forest litter, feces, beverages, pickles, etc.
Bacteria and archaea species with tannase activity have been found in the genera: Achromobacter, Atopobium, Azotobacter, Bacillus, Citrobacter, Corynebacterium, Enterobacter, Enterococcus, Fusobacterium, Gluconoacetobacter, Klebsiella, Lactobacillus, Lonepinella, Methanobrevibacter, Microbacterium, Oenococcus, Pantoea, Pediococcus, Providencia, Pseudomonas, Selenomonad, and Serratia.
In addition, some fungal species are dominant tannase producers, such as Aspergilli species.
Tannase is present in microorganisms, plants and animals.
However, microorganisms are mainly used for commercial production.
From the list of species of tannase producers given by them one can infer that interestingly fungi, yeasts and bacteria are the dominant groups among the microorganisms.
Among fungi, Aspergilli and Penicillia are the major groups although 20 different genera of fungi are known as tannase producers.
So far 27 species of Aspergillus, 24 species of Penicillium, 4 species of Trichoderma and 3 species of Fusarium are reported as tannase producers.
Among bacteria about 21 different genera are known as tannase producers and among them Lactobacilli are the dominant groups (13 species) followed by Pediococcus (4 species), Serratia, (3 species), Leuconostoc (2 species), Pantonea (2 species) Streptococcus (2 species) among others.
Early study focused on screening of microorganisms available as stock culture from culture collection centers which primarily derived microorganism from soil.
However, later investigators designed media specific for screening tannase producers from natural environment such as forest litter, human faeces, fermented foods, sheep excreta, tannery effluents, olive mill waste water, etc.
Tannase cleaves ester and depside linkages in such hydrolyzable tannins as tannic acid and chebulinic acid.
Tannase also acts on the ester and depside linkages in methylgallate and m-digallic acid, respectively.
Tannase hydrolyzes only those substrates that contain at least two phenolic OH groups in the acid component.
The esterified COOH group must be on the oxidized benzene ring and must not be ortho to one of the OH groups.
Tannase is a key enzyme in the degradation of gallotannins and ellagicitannins, two types of hydrolysable tannins.
KEYWORDS:
9025-71-2, 3.1.1.20, Tannin acyl Hydrolase, MFCD00212735, NA.54, powder, enzyme, 232-804-4, digallate, gallate
PROPERTIES of Tannase:
biological source:
Aspergillus sp. (A. ficuum)
form:
powder
specific activity:
≥150 U/g
impurities:
25 mM potassium phosphate
250 mM NaCl
50% glycerol
color:
white
storage temp.:
2-8°C
Tannases represent a group of enzymes finding its applications in food, brewing, and pharmaceutical industries.
They have a wide range of distribution and are reported form animals, plants, and microbial sources.
However, tannase from microbial source is preferred over other sources for industrial uses.
Tannases act upon hydrolyzable tannins by cleaving the ester and depside bonds so as to release glucose and gallic acid.
Gallic acid production is one of the most important commercial applications of tannase.
Apart from that, they are extensively used in the food industry, especially in instant tea production, where it enhances the extractability and cold water solubility of key compounds.
Another important application of tannase is the removal of haze formation and unflavored phenolic compounds from beer and wine.
Quality of fruit juices also can be improved by tannase enzyme.
Haze formation and bitterness of the fruit juices can be minimized by the application of these enzymes.
Tannins are considered as anti-nutritional factors while using agro-industrial residues as animal feed.
De-tannification of feed by tannase enzyme treatment can significantly improvise the quality of animal feed.
Synonym(s):
Tannin acyl Hydrolase
CAS Number:
9025-71-2
EC Number:
232-804-4
MDL number:
MFCD00212735
NACRES:
NA.54
Biochem/physiol Actions of Tannase:
Tannase catalyzes the hydrolysis of tannic acid to produce gallic acid and glucose.
Tannase is an enzyme that hydrolyzes Tannin containing Depside bonds such as Tannic acid and Chlorogenic acid.
Tannase is chemically identified as Tannin acylhydrolase with the following registry number: EC 3.1.1.20 and CAS RN: 9025-71-2.
Tannase is produced by a non-genetically modified Aspergillus oryzae strains, with the criteria laid out in Regulation (EC) 1332/2008 on food enzymes.
In the food industry, Tannase gives particularly excellent effects for improving tea quality (reduce bitterness) and preventing white clouding due to the coacervation of tea tannin.
Apart from the main enzymatic activities, Tannase does not contain significant levels of subsidiary activities.
Tannase is intended for use in the production of tea based beverages (ready to drink teas, tea extracts) and botanical extracts.
When using Tannase for other kinds of beverage, try with concentration between 0.01% and 0.2%, at 30 to 40℃ for a certain time period, to find out the optimum conditions.
Tannase should be noted that ferric ions as inhibitor may affect the result.
Tea based beverage:
- Improve tea quality (reduce bitterness) and prevent haze formation due to the coacervation of tea tannin
Tannase is added to tea extract at a concentration of about 0.005% to 0.2% according to the concentration of tannin and incubated at 30 to 40℃ for 0.5 to 1 hours under stirring.
- Increase extractability
Tannase and other enzyme preparations (e.g. Cellulase, Pectinase) are added to a tea/water slurry at a concentration of about 0.05% to 0.1% and incubated at 40℃ for 1 to 2 hours under stirring.
- Tannase is deactivated by heating at more than 95℃ for more than 30 min.
Botanical extract:
- Increase extractability
Tannase and other enzyme preparations (e.g. Cellulase, Pectinase) are added to botanical extract at a concentration of about 0.05% to 0.1% and incubated at 40℃ for 1 to 2 hours under stirring.
- Tannase is deactivated by heating at more than 95℃ for more than 30 min.
Tannase is an inducible enzyme used extensively in food, feed, pharmaceutical and chemical industries.
In this study, tannase production and its biochemical properties were evaluated.
From 42 Aspergillus strains analysed for potential tannase selection, Aspergillus melleus yielded the best results.
Production was analysed using a complete factorial planning of 2³.
Maximum activity (452.55 U mL−1) was obtained in the optimal conditions of substrate (5.0 g), initial moisture (60%), tannic acid (2%) and 48 h of fermentation.
The molecular weight of the purified enzyme was estimated as 69.52 kDa; its optimum temperature and pH were 40 °C and 5.5, respectively.
Regarding the chemical effectors used, tannase was inhibited by ZnCl2, ZnSO4, Triton X-100 and SDS.
The addition of tannase to green tea improved its antioxidant potential by approximately 85% when compared to the control.
The present results suggest that tannase may be used as an adjuvant to increase the antioxidant potential of green tea.
Tannase (EC 3.1.1.20) belongs to the class of hydrolases.
Tannase catalyzes the hydrolysis of digallate to gallate.
The systematic name of this enzyme class is tannin acylhydrolase.
Other names in common use include tannase S, and tannin acetylhydrolase.
Tannase is a natural adaptive intracellular/extracellular inducible hydrolase and placed in the esterase superfamily.
Tannase can be obtained from plants, animals and microorganisms, but the microbial-derived tannase is more extensive because its stability is higher than that of plant and animal sources.
Structure of Tannase:
Natural B. subtilis tannase consists of 9.3% α-helix, 33.6% parallel β-sheet, 17.2% β-turn and 39.9% random coil.
The β conformation plays a dominant role in tannase activity, and the secondary structure of tannase is stringently dependent on its microenvironment (temperature and pH).
B. Subtilis tannase scanning probe microscopyic analysis (SPM) showed that tannase showed different degrees of aggregation, the structure is similar to round or oval, the size is different, and the average diameter is 44 nm.
The crystal structure of tannase is a small plate-like crystal.
Three-dimensional structural analysis of L. plantarum tannase revealed that it exhibited α/β structure with 18 α-helices and 13 β-strands.
Physicochemical properties of Tannase:
The properties of tannase vary from species to species.
Tannase has a molecular weight of 46.5-90 kDa and exists as a monomer, while tannase from Rhodococcus sp. and L. plantarum contains two subunits.
So far, all tannases from yeast and fungi are glycoproteins, but there seems to be no such post-translational modification in bacteria.
Tannase is an acidic protein with an optimum pH range of 4.5-7.0.
The optimal temperature of different kinds of tannase is different, and the optimum temperature of most bacterial tannase is between 30 and 40 °C.
When methyl gallate was used as a substrate and the reaction temperature was 30-40 °C, the bacterial tannase substrate affinity (Km) from Selenomonas ruminantium and Enterobacter sp. was 1.6 and 3.7, respectively.
More than 28% of bacterial tannase requires metal ions as a cofactor to stimulate its maximum catalytic efficiency.
It has also been found that the activity of B. subtilis tannase is increased in polar protic solvents such as glycerol, isopropanol, ethanol, methanol and isoamyl alcohol, while butanol, acetic acid and acetone reduce the activity of tannase.
Tannase (Tan410) from a soil metagenomic library was immobilized on different supports, including mesoporous silica SBA-15, chitosan, calcium alginate, and amberlite IRC 50.
Entrapment in calcium alginate beads was comparatively found to be the best method and was further characterized.
The optimum pH of the immobilized Tan410 was shifted toward neutrality compared with the free enzyme (from pH 6.4 to pH 7.0).
The optimum temperature was determined to be 45°C for the immobilized enzyme and 30°C for the free enzyme, respectively.
The immobilized enzyme had no loss of activity after 10 cycles, and retained more than 90% of its original activity after storage for 30 days.
After immobilization, the enzyme activity was only slightly affected by Hg2+, which completely inhibited the activity of the free enzyme
The immobilized tannase was used to remove 80% of tannins from a green tea infusion on the first treatment.
The beads were used for six successive runs resulting in overall hydrolysis of 56% of the tannins.
Tannase (tannin acyl hydrolase, E.C.3.1.1.20) is an extracellular hydrolase enzyme that catalyzes the hydrolysis of ester and depside bonds in hydrolysable tannins or gallic acid esters, liberating glucose and gallic acid (GA).
Tannase cleaves the ester linkages between galloyl groups present in various compounds such as epigallocatechin and epigallocatechin gallate that are present in green tea leaves.
The enzyme could be obtained from many sources starting from prokaryotes to higher eukaryotes.
Vital minutiae such as regulation pathways, catalytic characteristics and other properties remain unrevealed which limits its usage in large scale.
This study essentially elicits the information on tannase substrates, mechanism, applications, and the recent trends in the purification of tannase.
Catalytic Mechanism of Tannase:
Tannase is the most studied enzyme in tannin biodegradation.
Tannase catalyzes the hydrolysis of esters and depside bonds of various substrates, including gallanonins, gallic acid esters, epigallocatechin gallate and epicatechin gallate, releasing gallic acid and glucose.
L. plantarum tannase consists of two domains, one α/β-hydrolase domain (residues 4-204 and 396-469) and a "lid" domain (residues 205-395).
The glycerol molecule in the cryoprotectant solution binds to the active site of the tannase, which mimick the binding of the galloyl moiety to its substrate.
The active site of tannase is located in the α/β-hydrolase domain, of which three amino acid residues Ser163, Asp419 and His451 are catalytic triads.
L. plantarum tannase contains 18 α-helices and 13 β-strands, and Ser163 is present in the Gly161-X-Ser163-X-Gly165 pentapeptide motif between β6 and α6.
In the active site, Ser163 forms a hydrogen bond with the NE2 atom of the His451 imidazole ring.
During the reaction, His451 deprotonated the hydroxyl group of Ser163, and Ser163 was stabilized by hydrogen bonding with Asp419.
Tannases are a family of esterases that catalyze the hydrolysis of ester and depside bonds present in hydrolyzable tannins to release gallic acid.
Here, a novel tannase from Lachnospiraceae bacterium (TanALb) was characterized.
The recombinant TanALb exhibited maximal activity at pH 7.0 and 50°C, and it maintained more than 70% relative activity from 30°C to 55°C.
The activity of TanALb was enhanced by Mg2+ and Ca2+, and was dramatically reduced by Cu2+ and Mn2+.
TanALb is capable of degrading esters of phenolic acids with long-chain alcohols, such as lauryl gallate as well as tannic acid.
The Km value and catalytic efficiency (kcat /Km) of TanALb toward five substrates showed that tannic acid (TA) was the favorite substrate.
Homology modeling and structural analysis indicated that TanALb contains an insertion loop (residues 341–450).
Based on the moleculer docking and molecular dynamics (MD) simulation, this loop was observed as a flap-like lid to interact with bulk substrates such as tannic acid.
TanALb is a novel bacterial tannase, and the characteristics of this enzyme make it potentially interesting for industrial use.
Tannins are polyphenolic compounds present in plants where they play an important role to prevent the attack of viruses, bacteria and fungi.
Despite the fact that polyphenols inhibit the microbial growth, adaptation process has allowed developing mechanisms to transform them.
One mechanism is the production of tannase, which has been obtained mostly from fungi.
In recent years, some tannase producer bacteria have been isolated from different sources, mainly from animals and human intestine and feces as well as from fermented food and fruit wastes.
Obtaining high titers of bacterial tannase depends mainly on the culture medium composition, the bacterial strain and the process optimization of culture conditions.
This paper presents an overview of the recent investigations regarding the production, the physicochemical and molecular characteristics, the applications and the potential uses of bacterial tannases.
Tannase is an enzyme that hydrolyzes esters and lateral bonds of tannins, such as tannic acid, releasing glucose and gallic acid and stands out in the clarification of wines and juices.
Fungi of the genera Aspergillus and Penicillium are excellent producers of this enzyme.
The search for fungi that produce high levels of tannase as well as new substrates for the enzyme production by the SSF is required.
The objectives of this study were to evaluate the production of tannase by Aspergillus and Penicillium species through SSF using leaves and agroindustrial waste barbados cherry and mangaba fruit as substrate, select the best producer, optimize production, characterize the crude enzyme extract, and apply it the clarification of grape juice.
Selecting the best producer was performed by planning Placket-Burman and RSM. P. montanense showed highest activity with 41.64 U/mL after 72 h of fermentation residue using barbados cherry, with 3.5% tannic acid and 70% moisture.
The enzyme showed the highest activity at pH 9.0 and 50°C.
The tannase of P. montanense was stable over a wide pH range and temperature and, when applied to grape juice, showed higher efficiency by reducing 46% of the tannin content after incubation 120 m.